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The aim of this study was to evaluate the preclinical efficacy of [ 89 Zr]Zr-DFO-Ab253 as a novel positron emission tomography (PET) tracer for CD146-positive malignant melanoma imaging. Considering the high expression of CD146 in malignant melanoma, this study investigated the effect of different CD146 expression levels on the tumor uptake of [ 89 Zr]Zr-DFO-Ab253. CD146 selectivity was investigated by using the CD146-positive human melanoma cell A375 and the CD146-negative human alveolar epithelial cell A549. The cell uptake of [ 89 Zr]Zr-DFO-Ab253 tracers was investigated, and receptor-binding affinities were measured by radioactive enzyme-linked immunosorbent assay. Biodistribution studies and micro-PET imaging of the radiotracers were performed on mice bearing A375 and A549 xenografts under baseline and blocking conditions. An immunohistochemical test was performed using A375 and A549 tissue sections for CD146 expression level analysis. [ 89 Zr]Zr-DFO-Ab253 was obtained with a high radiochemical yield (87.86 ± 4.66%) and a satisfactory radiochemical purity (>98.0%). The specificity and affinity of [ 89 Zr]Zr-DFO-Ab253 were confirmed in melanoma A375 cells and in vivo PET imaging of A375 tumor models. [ 89 Zr]Zr-DFO-IgG and A549 lung tumors were prepared as control radiotracers and negative models to verify the specificity of [ 89 Zr]Zr-DFO-Ab253 on CD146. [ 89 Zr]Zr-DFO-Ab253 has a K d of 4.01 ± 0.50 nM. PET imaging and biodistribution showed a higher uptake of [ 89 Zr]Zr-DFO-Ab253 in A375 melanomas than that in A549 tumors (42.1 ± 4.04% vs 7.87 ± 1.30% ID/g at 120 h, P < 0.05). A low tumor uptake of [ 89 Zr]Zr-DFO-IgG was observed with uptakes of 1.91 ± 0.41 and 2.80 ± 0.14 ID%/g when blocked at 120 h. The radiation-absorbed dose was calculated to be 0.13 mSv/MBq. This study demonstrates the synthesis and preclinical evaluation of [ 89 Zr]Zr-DFO-Ab253 and indicates that the novel tracer has promising applications in malignant melanoma-specific PET imaging because of its high uptake and long-time retention in malignant melanoma. It also provides feasibility for the development of integrated molecular probes for diagnosis and treatment based on the CD146 target.

The perivascular localization of endometrial mesenchymal stem/stromal cells (eMSC) allows them to sense local and distant tissue damage, promoting tissue repair and healing. Our hypothesis is that eMSC therapeutic effects are largely exerted via their exosomal secretome (eMSC EXOs) by targeting the immune system and angiogenic modulation. For this purpose, EXOs isolated from Crude and CD146+ eMSC populations were compared for their miRNA therapeutic signatures and immunomodulatory functionality under inflammatory conditions. eMSC EXOs profiling revealed 121 in Crude and 88 in CD146+ miRNAs, with 82 commonly present in both populations. Reactome and KEGG analysis of miRNAs highly present in eMSC EXOs indicated their involvement among others in immune system regulation. From the commonly present miRNAs, four miRNAs (hsa-miR-320e, hsa-miR-182-3p, hsa-miR-378g, hsa-let-7e-5p) were more enriched in CD146+ eMSC EXOs. These miRNAs are involved in macrophage polarization, T cell activation, and regulation of inflammatory cytokine transcription (i.e., TNF-α, IL-1β, and IL-6). Functionally, stimulated macrophages exposed to eMSC EXOs demonstrated a switch towards an alternate M2 status and reduced phagocytic capacity compared to stimulated alone. However, eMSC EXOs did not suppress stimulated human peripheral blood mononuclear cell proliferation, but significantly reduced secretion of 13 pro-inflammatory molecules compared to stimulated alone. In parallel, two anti-inflammatory proteins, IL-10 and IL-13, showed higher secretion, especially upon CD146+ eMSC EXO exposure. Our study suggests that eMSC, and even more, the CD146+ subpopulation, possess exosomal secretomes with strong immunomodulatory miRNA attributes. The resulting evidence could serve as a foundation for eMSC EXO-based therapeutics for the resolution of detrimental aspects of tissue inflammation.

Purpose: Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) proteins and leads to the maladaptive changes in myocardium. Endothelial cells (ECs) undergoing mesenchymal transition contributes to the occurrence and development of cardiac fibrosis. CD146 is an adhesion molecule highly expressed in ECs. The present study was performed to explore the role of CD146 in modulating endothelial to mesenchymal transition (EndMT)., Methods: C57BL/6 mice were subjected to subcutaneous implantation of osmotic minipump infused with angiotensin II (Ang Ⅱ). Adenovirus carrying CD146 short hairpin RNA (shRNA) or CD146 encoding sequence were infected into cultured human umbilical vein endothelial cells (HUVECs) followed by stimulation with Ang II or transforming growth factor-β1 (TGF-β1). Differentially expressed genes were revealed by RNA-sequencing (RNA-Seq) analysis. Gene expression was measured by quantitative real-time PCR, and protein expression and distribution were determined by Western blot and immunofluorescence staining, respectively., Results: CD146 was predominantly expressed by ECs in normal mouse hearts. CD146 was upregulated in ECs but not fibroblasts and myocytes in hearts of Ang II-infused mice and in HUVECs stimulated with Ang Ⅱ. RNA-Seq analysis revealed the differentially expressed genes related to EndMT and Wnt/β-catenin signaling pathway. CD146 knockdown and overexpression facilitated and attenuated, respectively, EndMT induced by Ang II or TGF-β1. CD146 knockdown upregulated Wnt pathway-related genes including Wnt4, LEF1, HNF4A, FOXA1, SOX6, and CCND3, and increased the protein level and nuclear translocation of β-catenin., Conclusions: Knockdown of CD146 exerts promotional effects on EndMT via activating Wnt/β-catenin pathway and the upregulation of CD146 might play a protective role against EndMT and cardiac fibrosis., Competing Interests: The authors have declared that no competing interests exist.

Initiation of T cell receptor (TCR) signaling involves the activation of the tyrosine kinase LCK; however, it is currently unclear how LCK is recruited and activated. Here, we have identified the membrane protein CD146 as an essential member of the TCR network for LCK activation. CD146 deficiency in T cells substantially impaired thymocyte development and peripheral activation, both of which depend on TCR signaling. CD146 was found to directly interact with the SH3 domain of coreceptor-free LCK via its cytoplasmic domain. Interestingly, we found CD146 to be present in both monomeric and dimeric forms in T cells, with the dimerized form increasing after TCR ligation. Increased dimerized CD146 recruited LCK and promoted LCK autophosphorylation. In tumor models, CD146 deficiency dramatically impaired the antitumor response of T cells. Together, our data reveal an LCK activation mechanism for TCR initiation. We also underscore a rational intervention based on CD146 for tumor immunotherapy.

Multiple sclerosis is a chronic auto-inflammatory disease of the central nervous system affecting patients worldwide. Neuroinflammation in multiple sclerosis is mainly driven by peripheral immune cells which invade the central nervous system and cause neurodegenerative inflammation. To enter the target tissue, immune cells have to overcome the endothelium and transmigrate into the tissue. Numerous molecules mediate this process and, as they determine the tissue invasiveness of immune cells, display great therapeutic potential. Melanoma cell adhesion molecule (MCAM) is a membrane-anchored glycoprotein expressed by a subset of T-cells and MCAM+ T-cells have been shown to contribute to neuroinflammation in multiple sclerosis. The role of the MCAM molecule for brain invasion, however, remained largely unknown. In order to investigate the role of the MCAM molecule on T-cells, we used different in vitro and in vivo assays, including ex vivo flow chambers, biochemistry and microscopy experiments of the mouse brain. We demonstrate that MCAM directly mediates adhesion and that the engagement of MCAM induces intracellular signaling leading to β1-integrin activation on human T-cells. Furthermore, we show that MCAM engagement triggers the phosphorylation of PLCγ1 which is required for integrin activation and thus amplification of the cellular adhesive potential. To confirm the physiological relevance of our findings in vivo , we demonstrate that MCAM plays an important role in T-cell recruitment into the mouse brain. In conclusion, our data demonstrate that MCAM expressed on T-cells acts as an adhesion molecule and a signaling receptor that may trigger β1-integrin activation via PLCγ1 upon engagement., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Zondler, Herich, Kotte, Körner, Schneider-Hohendorf, Wiendl, Schwab and Zarbock.)

Background: MUC18 is a glycoprotein highly expressed on the surface of melanoma and other cancers which promotes tumor progression and metastasis. However, its mechanism of action and suitability as a therapeutic target are unknown., Methods: A monoclonal antibody (mAb) (JM1-24-3) was generated from metastatic melanoma tumor live cell immunization, and high-throughput screening identified MUC18 as the target., Results: Analysis of molecular interactions between MUC18 and JM1-24-3 revealed that the downstream signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18. JM1-24-3 inhibited melanoma cell proliferation, migration and invasion in vitro and reduced tumor growth and metastasis in vivo., Conclusion: These results confirm that MUC18 is mechanistically important in melanoma growth and metastasis, suggest that the MUC18 epitope identified is a promising therapeutic target, and that the JM1-24-3 mAb may serve as the basis for a potential therapeutic agent.

In this study we used two different techniques in order to isolate pericytes from the wall of human umbilical cord vein and get two different groups of cells were named as "pellet and primer cells". These groups were compared with each other according to their morphologies and stem cell marker expressions. Also, these two different populations were compared with each other and with human bone marrow mesenchymal stem cells (BM-MSCs) according to their transcriptomic profiles. Then, pellet cells proteomic profiles were determined. Our results showed that morphologies and cell surface marker expressions of pellet cells and primer cells are similar. On the other hand, according to immunofluorescence staining results, in contrast to primer cells, pellet cells showed positive NG2 and PDGFR-β staining. As a result of gene expression profiling, pellet cells have upregulated genes related with muscle, neural and immune cell differentiation, development and pluripotency. On the other hand, primer cells have upregulated adhesion pathway-related genes. In addition to differences between pellet and primer cells, the gene expression profiles of these cell groups are also different from BM-MSCs. The results of transcriptome and proteome analysis of pellet cells were in consistent with each other.

Our understanding of mesenchymal cell subsets and their function in human lung affected by aging and in certain disease settings remains poorly described. We use a combination of flow cytometry, prospective cell-sorting strategies, confocal imaging, and modeling of microvessel formation using advanced microfluidic chip technology to characterize mesenchymal cell subtypes in human postnatal and adult lung. Tissue was obtained from patients undergoing elective surgery for congenital pulmonary airway malformations (CPAM) and other airway abnormalities including chronic obstructive pulmonary disease (COPD). In microscopically normal postnatal human lung, there was a fivefold higher mesenchymal compared with epithelial (EpCAM + ) fraction, which diminished with age. The mesenchymal fraction composed of CD90 + and CD90 + CD73 + cells was enriched in CXCL12 and platelet-derived growth factor receptor-α (PDGFRα) and located in close proximity to EpCAM + cells in the alveolar region. Surprisingly, alveolar organoids generated from EpCAM + cells supported by CD90 + subset were immature and displayed dysplastic features. In congenital lung lesions, cystic air spaces and dysplastic alveolar regions were marked with an underlying thick interstitium composed of CD90 + and CD90 + PDGFRα + cells. In postnatal lung, a subset of CD90 + cells coexpresses the pericyte marker CD146 and supports self-assembly of perfusable microvessels. CD90 + CD146 + cells from COPD patients fail to support microvessel formation due to fibrinolysis. Targeting the plasmin-plasminogen system during microvessel self-assembly prevented fibrin gel degradation, but microvessels were narrower and excessive contraction blocked perfusion. These data provide important new information regarding the immunophenotypic identity of key mesenchymal lineages and their change in a diverse setting of congenital lung lesions and COPD.

Antibody biorecognition forms the basis for numerous biomedical applications such as diagnostic assays, targeted drug delivery, and targeted cancer imaging. However, antibodies, especially after being conjugated to surfaces or nanostructures, suffer from stability issues when stored under nonrefrigeration conditions. Therefore, enhancing the stability of antibodies on surfaces and nanostructures under ambient and elevated temperatures is of paramount importance for many nanobiotechnology applications. In this study, we introduce a simple and facile approach based on a metal-organic framework (MOF) coating to preserve the biorecognition capability of antibodies immobilized on nanoscale surfaces after exposure to elevated temperatures for a prolonged period. By using atomic force microscopy (AFM)-based force spectroscopy, we demonstrate that the MOF coating is able to preserve the binding force and binding frequency of the anti-CD-146 antibody attached to an AFM tip to CD-146 antigen on the surface of melanoma cells at the single-molecule level. We also demonstrate that the MOF coating outperforms another commonly used sucrose coatings in terms of maintaining the binding force and binding frequency of the antibody to antigen. Herein, the AFM tip functionalized with antibodies provides a nanoscale testbed (analogous to an antibody-conjugated nanostructure) to assess antibody biorecognition at the single-molecule level and preservation efficacy under antibody denaturing conditions. This MOF coating approach should be applicable to the preservation of a variety of antibody-conjugated nanostructures aiming for targeted drug delivery, targeted cancer imaging, and nanobiosensors. The improved stability and elimination of refrigeration requirements will facilitate wide applications of antibody-enabled nanobiotechnology in resource-limited environments and populations.

Tumor endothelial cells (TECs), which constitute the lining of the tumor blood vessels, have various characteristics as tumor constituent cells. In this study, we describe a novel method for the isolation of highly pure, fresh TECs, which form a small population within the tumor. Tumors were first dissected from tumor-bearing mice and digested to a single cell suspension with Collagenase Type II; the single cells were then separated by density gradient centrifugation. TECs were enriched by CD31-positive selection using magnetic activated cell sorting and subsequently purified by fluorescence activated cell sorting. The high purity of the obtained cells was verified by flow cytometry. Upon cell culture, the isolated cells showed a polygonal shape and a cobblestone appearance, which are features of the endothelial cells. Furthermore, a functional assay revealed that the TECs suppressed the proliferation of CD8+ T cells in vitro. We believe that the isolation method described in this study will enable the further elucidation of the characteristics of TECs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Very late antigen 4 (VLA-4; integrin α4β1) is critical for transmigration of T helper (T H ) 1 cells into the central nervous system (CNS) under inflammatory conditions such as multiple sclerosis (MS). We have previously shown that VLA-4 and melanoma cell adhesion molecule (MCAM) are important for trans-endothelial migration of human T H 17 cells in vitro and here investigate their contribution to pathogenic CNS inflammation., Methods: Antibody blockade of VLA-4 and MCAM is assessed in murine models of CNS inflammation in conjunction with conditional ablation of α4-integrin expression in T cells. Effects of VLA-4 and MCAM blockade on lymphocyte migration are further investigated in the human system via in vitro T cell transmigration assays., Results: Compared to the broad effects of VLA-4 blockade on encephalitogenic T cell migration over endothelial barriers, MCAM blockade impeded encephalitogenic T cell migration in murine models of MS that especially depend on CNS migration across the choroid plexus (CP). In transgenic mice lacking T cell α4-integrin expression (CD4::Itga4 -/- ), MCAM blockade delayed disease onset. Migration of MCAM-expressing T cells through the CP into the CNS was restricted, where laminin 411 (composed of α4, β1, γ1 chains), the proposed major ligand of MCAM, is detected in the endothelial basement membranes of murine CP tissue. This finding was translated to the human system; blockade of MCAM with a therapeutic antibody reduced in vitro transmigration of MCAM-expressing T cells across a human fibroblast-derived extracellular matrix layer and a brain-derived endothelial monolayer, both expressing laminin α4. Laminin α4 was further detected in situ in CP endothelial-basement membranes in MS patients' brain tissue., Conclusions: Our findings suggest that MCAM-laminin 411 interactions facilitate trans-endothelial migration of MCAM-expressing T cells into the CNS, which seems to be highly relevant to migration via the CP and to potential future clinical applications in neuroinflammatory disorders.

Extracellular matrix (ECM) proteins secreted by blood-brain barrier (BBB) endothelial cells (ECs) are implicated in cell trafficking. We discovered that the expression of ECM epidermal growth factor-like protein 7 (EGFL7) is increased in the CNS vasculature of patients with multiple sclerosis (MS), and in mice with experimental autoimmune encephalomyelitis (EAE). Perivascular CD4 T lymphocytes colocalize with ECM-bound EGFL7 in MS lesions. Human and mouse activated T cells upregulate EGFL7 ligand αvβ3 integrin and can adhere to EGFL7 through integrin αvβ3. EGFL7-knockout (KO) mice show earlier onset of EAE and increased brain and spinal cord parenchymal infiltration of T lymphocytes. Importantly, EC-restricted EGFL7-KO is associated with a similar EAE worsening. Finally, treatment with recombinant EGFL7 improves EAE, reduces MCAM expression, and tightens the BBB in mouse. Our data demonstrate that EGFL7 can limit CNS immune infiltration and may represent a novel therapeutic avenue in MS.

CD146 has been identified as an excellent biomarker for lung cancer as its overexpression in solid tumors has been linked to disease progression, invasion, and metastasis. Previously, our group described a positive correlation between 64 Cu-labeled YY146 uptake and increased expression of CD146 in six human lung cancer cell lines using subcutaneous tumor models. In this study, we investigate a monoclonal antibody called YY146 for immunoPET imaging of CD146 in two intrapulmonary metastasis models of non-small cell lung cancer (NSCLC). The binding and immunoreactivity of the tracer were assessed by in vitro assays. Radiolabeling of YY146 with positron emitting Cu-64 ( 64 Cu-NOTA-YY146) enabled PET imaging of intrapulmonary metastasis. Mice were intravenously injected with two million tumor cells, and CT imaging was used to verify the presence of lung metastases. 64 Cu-NOTA-YY146 was injected into tumor-bearing mice, and animals were subjected to PET/CT imaging at 4, 24, and 48 h postinjection. Both the average and maximum lung PET signal intensities were quantified and compared between high and low CD146-expressing metastases. Further validation was accomplished through immunofluorescence imaging of resected tissues with CD31 and CD146. In flow cytometry, YY146 revealed strong binding to CD146 in H460 cells due to its high expression with minimal binding to CD146-low expressing H358 cells. Both YY146 and NOTA-YY146 showed similar binding, suggesting that NOTA conjugation did not elicit any negative effects on its binding affinity. Imaging of 64 Cu-NOTA-YY146 in H460 tumor-bearing mice revealed rapid, persistent, and highly specific tracer accumulation. Uptake of 64 Cu-NOTA-YY146 in the whole lung was calculated for H460 and H358 as 7.43 ± 0.38 and 3.95 ± 0.47% ID/g at 48 h postinjection (n = 4, p < 0.05), and the maximum lung signals were determined to be 13.85 ± 1.07 (H460) and 6.08 ± 0.73% ID/g (H358) at equivalent time points (n = 4, p < 0.05). To ensure the specificity of the tracer, a nonspecific antibody was injected into H460 tumor-bearing mice. Ex vivo biodistribution and immunofluorescence imaging validated the PET findings. In summary, 64 Cu-NOTA-YY146 allowed for successful imaging of CD146-expressing intrapulmonary metastases of NSCLC in mice. This preliminary study provides evidence supporting the future clinical utilization of 64 Cu-NOTA-YY146 for possible treatment monitoring of CD146-targeted therapy or improving patient stratification.

Objective: To evaluate the effects of MUC18 on IL-13-mediated airway inflammatory responses in human airway epithelial cells and in mice., Materials: Primary normal human tracheobronchial epithelial (HTBE) cells, wild-type (WT) and Muc18 knockout (KO) mice, and mouse tracheal epithelial cells (mTECs) were utilized., Treatment: Cultured HTBE cells treated with MUC18 siRNA or MUC18 expressing lentivirus were incubated with IL-13 (10 ng/mL) for 24 h. Mice were intranasally instilled with 500 ng of IL-13 for 3 days. mTECs were treated with IL-13 (10 ng/mL) for 3 days., Methods: PCR was used to measure mRNA expression. Western Blot and ELISAs were used to quantify protein expression. Cytospins of bronchoalveolar lavage (BAL) cells were used to obtain leukocyte differentials., Results: MUC18 siRNA reduced IL-13-mediated eotaxin-3 (183 ± 44 vs. 380 ± 59 pg/mL, p < 0.05), while MUC18 overexpression increased IL-13-mediated eotaxin-3 (95 ± 3 vs. 58 ± 3 pg/mL, p < 0.05) in HTBE cells. IL-13-treated Muc18 KO mice had a lower percentage of neutrophils in BAL than WT mice (25 ± 3 vs. 35 ± 3%, p = 0.0565)., Conclusions: These results implicate MUC18 as a potential enhancer of airway inflammation in a type 2 cytokine (e.g., IL-13) milieu.

Objective: Intrinsic inflammatory characteristics play a pivotal role in stem cell recruitment and homing through migration where the subsequent change in niche has been shown to alter these characteristics. The bone marrow mesenchymal stem cells (bmMSCs) have been demonstrated to migrate to the endometrium contributing to the stem cell reservoir and regeneration of endometrial tissue. Thus, the aim of the present study was to compare the inflammation-driven migration and cytokine secretion profile of human bmMSCs to endometrial mesenchymal stem cells (eMSCs) and endometrial fibroblasts (eSFs)., Materials and Methods: The bmMSCs were isolated from bone marrow aspirates through culturing, whereas eMSCs and eSFs were FACS-isolated. All cell types were tested for their surface marker, proliferation profiles and migration properties towards serum and inflammatory attractants. The cytokine/chemokine secretion profile of 35 targets was analysed in each cell type at basal level along with lipopolysaccharide (LPS)-induced state., Results: Both stem cell types, bmMSCs and eMSCs, presented with similar stem cell surface marker profiles as well as possessed high proliferation and migration potential compared to eSFs. In multiplex assays, the secretion of 16 cytokine targets was detected and LPS stimulation expanded the cytokine secretion pattern by triggering the secretion of several targets. The bmMSCs exhibited higher cytokine secretion of vascular endothelial growth factor (VEGF)-A, stromal cell-derived factor-1 alpha (SDF)-1α, interleukin-1 receptor antagonist (IL-1RA), IL-6, interferon-gamma inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)1α and RANTES compared to eMSCs and/or eSFs after stimulation with LPS. The basal IL-8 secretion was higher in both endometrial cell types compared to bmMSCs., Conclusion: Our results highlight that similar to bmMSCs, the eMSCs possess high migration activity while the differentiation process towards stromal fibroblasts seemed to result in loss of stem cell surface markers, minimal migration activity and a subtler cytokine profile likely contributing to normal endometrial function.

Although progress was made in in vitro fertilization (IVF) techniques, the majority of embryos transferred fail to implant. Morphology embryo scoring is the standard procedure for most of IVF centres for choosing the best embryo, but remains limited since even the embryos classified as "top quality" may not implant. As it has been shown that i) CD146 is involved in embryo implantation and ii) membrane form is shed to generate soluble CD146 (sCD146), we propose that sCD146 in embryo supernatants may constitute a new biomarker of embryo selection. Immunocytochemical staining showed expression of CD146 in early embryo stages and sCD146 was detected by ELISA and Western-blot in embryo supernatants from D2. We retrospectively studied 126 couples who underwent IVF attempt. The embryo culture medium from each transferred embryo (n = 222) was collected for measurement of sCD146 by ELISA. Significantly higher sCD146 concentrations were present in embryo supernatants that did not implant (n = 185) as compared to those that successfully implanted (n = 37) (1310 +/- 1152 pg.mL-1 vs. 845+/- 1173 pg.mL-1, p = 0.024). Sensitivity analysis performed on single embryo transfers (n = 71) confirmed this association (p = 0.0054). The computed ROC curve established that the optimal sCD146 concentration for embryo implantation is under 1164 pg.mL-1 (sensitivity: 76%, specificity: 48%, PPV: 25% and NPV: 92%). Over this sCD146 threshold, the implantation rate was significantly lower (9% with sCD146 levels >1164 pg.ml-1 vs. 22% with sCD146 levels ≤ 1164 pg.mL-1, p = 0.01). Among the embryos preselected by morphologic scoring, sCD146 determination could allow a better selection of the embryo(s), thus improving the success of elective single embryo transfer. This study establishes the proof of concept for the use of sCD146 as a biomarker for IVF by excluding the embryo with the highest sCD146 level. A multicentre prospective study will now be necessary to further establish its use in clinical practice.

Breast cancer is the most common malignancy in women. Altered expression of MUC18, a cell surface receptor, and its interaction with Wnt-5a as its ligand, affects the motility and invasiveness of breast cancer cells. In this study, we explored the Wnt-5a binding site and designed an antigenic epitope on the MUC18 receptor using in silico methods. A specific single-chain variable fragment (scFv) was isolated against the epitope by several panning processes. The binding ability of the scFv to the related epitope was evaluated in ELISA and flow cytometry. The inhibitory effects of the selected scFv on MUC18 positive cell line, MDA-MB231, was assessed by migration and invasion assays. The results demonstrated isolation of specific scFv with frequency of 40 % which showed significant binding with the epitope in both ELISA and fluorescence-activated cell sorting (FACS) analyses. The antibody inhibited the migration (76 %) and invasion (67 %) of MUC18 positive cell line. The results suggest the specific anti-MUC18 scFv as an effective antibody for breast cancer immunotherapy.

Purpose: Overexpression of CD146 in solid tumors has been linked to disease progression, invasion, and metastasis. We describe the generation of a 64 Cu-labeled CD146-specific antibody and its use for quantitative immunoPET imaging of CD146 expression in six lung cancer models., Methods: The anti-CD146 antibody (YY146) was conjugated to 1,4,7-triazacyclononane-triacetic acid (NOTA) and radiolabeled with 64 Cu. CD146 expression was evaluated in six human lung cancer cell lines (A549, NCI-H358, NCI-H522, HCC4006, H23, and NCI-H460) by flow cytometry and quantitative western blot studies. The biodistribution and tumor uptake of 64 Cu-NOTA-YY146 was assessed by sequential PET imaging in athymic nude mice bearing subcutaneous lung cancer xenografts. The correlation between CD146 expression and tumor uptake of 64 Cu-NOTA-YY146 was evaluated by graphical software while ex vivo biodistribution and immunohistochemistry studies were performed to validate the accuracy of PET data and spatial expression of CD146., Results: Flow cytometry and western blot studies showed similar findings with H460 and H23 cells showing high levels of expression of CD146. Small differences in CD146 expression levels were found among A549, H4006, H522, and H358 cells. Tumor uptake of 64 Cu-NOTA-YY146 was highest in CD146-expressing H460 and H23 tumors, peaking at 20.1 ± 2.86 and 11.6 ± 2.34 %ID/g at 48 h after injection (n = 4). Tumor uptake was lowest in the H522 model (4.1 ± 0.98 %ID/g at 48 h after injection; n = 4), while H4006, A549 and H358 exhibited similar uptake of 64 Cu-NOTA-YY146. A positive correlation was found between tumor uptake of 64 Cu-NOTA-YY146 (%ID/g) and relative CD146 expression (r 2  = 0.98, p < 0.01). Ex vivo biodistribution confirmed the accuracy of the PET data., Conclusion: The strong correlation between tumor uptake of 64 Cu-NOTA-YY146 and CD146 expression demonstrates the potential use of this radiotracer for imaging tumors that elicit varying levels of CD146. In the future, this tool may promote enhanced monitoring of therapeutic response and improved patient stratification.

Background: Osteosarcoma is a rare form of cancer but with a substantial need for new active drugs. There is a particular need for targeted therapies to combat metastatic disease. One possible approach is to use an antibody drug conjugate or an antibody radionuclide conjugate to target the osteosarcoma metastases and circulating tumor cells. Herein we have evaluated a radiolabeled monoclonal antibody targeting CD146 both in vitro and in vivo., Methods and Results: A murine monoclonal anti-CD146 IgG1 isotype antibody, named OI-3, was developed along with recombinant chimeric versions with human IgG1 or human IgG3 Fc sequences. Using flow cytometry, selective binding of OI-3 to human osteosarcoma cell lines OHS, KPDX and Saos-2 was confirmed. The results confirm a higher expression level of CD146 on human osteosarcoma cells than HER2 and EGFR; antigens targeted by commercially available therapeutic antibodies. The biodistribution of 125I-labeled OI-3 antibody variants was compared with 125I-labeled chimeric anti-EGFR antibody cetuximab in nude mice with subcutaneous OHS osteosarcoma xenografts. OI-3 was able to target CD146 expressing tumors in vivo and showed improved tumor to tissue targeting ratios compared with cetuximab. Subsequently, the three OI-3 variants were conjugated with p-SCN-Bn-DOTA and labeled with a more therapeutically relevant radionuclide, 177Lu, and their biodistributions were studied in the nude mouse model. The 177Lu-labeled OI-3 variants were stable and had therapeutically relevant biodistribution profiles. Dosimetry estimates showed higher absorbed radiation dose to tumor than all other tissues after administration of the chimeric IgG1 OI-3 variant., Conclusion: Our results indicate that CD146 can be targeted in vivo by the radiolabeled OI-3 antibodies., Competing Interests: Competing Interests: The OI-3 monoclonal antibody is a patented product from Oncoinvent AS. Sara Westrøm and Tina B. Bønsdorff are employed by Oncoinvent AS, and Nasir Abbas and Thora J. Jonasdottir were employed by Oncoinvent AS at the time when their main contribution to the research article occurred. Oncoinvent AS provided support in the form of salaries for authors SW, TBB, NA and TJJ, but did not have any additional role in the study design, data collection and analysis, or preparation of the manuscript. Øyvind S. Bruland, Thora J. Jonasdottir and Roy H. Larsen are members of the Board of Oncoinvent AS, and the three of them together with Tina B. Bønsdorff are shareholders of Oncoinvent AS. Roy H. Larsen is also affiliated to Sciencons AS which is a shareholder of Oncoinvent AS. Sciencons AS did not provide support in the form of salaries for author Roy H. Larsen, and did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. A patent application related to the OI-3 antibody versions described in the submitted manuscript has previously been filed (Patent name: Monoclonal antibody and derivatives. Patent number: PCT/EP2014/070395). The authors confirm that these competing interests do not alter their adherence to PLOS ONE policies on sharing data and materials.

CD146 (MUC-18, MCAM) expression on cancer cells correlates with cancer progression and a bad prognosis in several tumors, including melanoma and pancreatic tumors. Deciphering the mechanism mediating the CD146 role in cancer is essential for generating new therapeutic strategies. We found that CD146 expression in cancer cells is associated with a secretion of soluble CD146 (sCD146) that constitutes an active player in tumor development. Indeed, sCD146 induces the overexpression of its binding protein, angiomotin, on both endothelial and cancer cells and promotes both paracrine effects on angiogenesis and autocrine effects on cancer cells proliferation and survival. These last effects are mediated in part through the induction and phosphorylation of c-myc in cancer cells. In mice models xenografted with human CD146-positive melanoma or pancreatic cancer cells, administration of a novel monoclonal antibody specifically targeting sCD146, but not its membrane form, successfully suppresses tumor vascularization and growth. Our findings demonstrate that sCD146 secreted by CD146-positive tumors mediates important pro-angiogenic and pro-tumoral effects. Targeting sCD146 with a novel neutralizing antibody could thus constitute an innovative therapeutic strategy for the treatment of CD146-positive tumors.

BACKGROUND Cervical cancer is one of the major causes of cancer death of females worldwide. Radiotherapy is considered effective for cervical cancer treatment, but the low radiosensitivity found in some cases severely affects therapeutic outcomes. This study aimed to reveal the role of CD146, an important adhesion molecule facilitating tumor angiogenesis, in regulating radiosensitivity of cervical cancer cells. MATERIAL AND METHODS CD146 protein expression was compared in normal cells, cervical cancer cells with lower radiosensitivity, and cervical cancer cells with higher sensitivity from cervical squamous cell carcinoma patients. Anti-CD146 monoclonal antibody AA98 was used to inhibit CD146 in human cervical cancer SiHa cells with relatively low radiosensitivity, and then the cell survival and apoptosis changes after radiation were detected by colony formation assay and flow cytometry. RESULTS CD146 protein was significantly up-regulated in cervical cancer cells (P<0.001), especially in cancer cells with lower radiosensitivity. The SiHa cells treated with AA98 showed more obvious inhibition in cell survival (P<0.05) and promotion in cell apoptosis (P<0.01) after radiation, compared to the untreated cells. More dramatic changes in apoptotic factors Caspase 3 and Bcl-XL were also detected in AA98-treated cells. CONCLUSIONS These results indicate that inhibiting CD146 improves the effect of radiation in suppressing SiHa cells. This study shows the potential of CD146 as a target for increasing radiosensitivity of cervical cancer cells, which might allow improvement in treatment outcome in cervical cancer. Further studies are necessary for understanding the detailed mechanism of CD146 in regulating radiosensitivity.

In utero hematopoietic stem/progenitor cell transplantation (IUHSCT) has only been fully successful in the treatment of congenital immunodeficiency diseases. Using sheep as a large animal model of IUHSCT, we demonstrate that administration of CD146(+)CXCL12(+)VEGFR2(+) or CD146(+)CXCL12(+)VEGFR2(-) cells prior to, or in combination with, hematopoietic stem/progenitor cells (HSC), results in robust CXCL12 production within the fetal marrow environment, and significantly increases the levels of hematopoietic engraftment. While in the fetal recipient, donor-derived HSC were found to reside within the trabecular bone, the increased expression of VEGFR2 in the microvasculature of CD146(+)CXCL12(+)VEGFR2(+) transplanted animals enhanced levels of donor-derived hematopoietic cells in circulation. These studies provide important insights into IUHSCT biology, and demonstrate the feasibility of enhancing HSC engraftment to levels that would likely be therapeutic in many candidate diseases for IUHSCT., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice., (© 2015 International Society for Advancement of Cytometry.)

The endothelial phenotype of tumor blood vessels differs from the liver and forms an important base for endothelium-specific targeting by antibody-coated nanoparticles. Although differences of shear stress and ligand avidity can modulate the nanoparticle binding to endothelium, these mechanisms are still poorly studied. This study analyzed the binding of antibody-coated nanoparticles to tumor and liver endothelium under controlled flow conditions and verified this binding in tumor models in vivo. Binding of anti-CD146-coated nanoparticles, but not of antibody was significantly reduced under increased wall shear stress and the degree of nanoparticle binding correlated with the avidity of the coating. The intravascular wall shear stress favors nanoparticle binding at the site of higher avidity of endothelial epitope which additionally promotes the selectivity to tumor endothelium. After intravenous application in vivo, pegylated self-coated nanoparticles showed specific binding to tumor endothelium, whereas the nanoparticle binding to the liver endothelium was very low. This study provides a rationale that selective binding of mAb-coated nanoparticles to tumor endothelium is achieved by two factors: higher expression of endothelial epitope and higher nanoparticle shearing from liver endothelium. The combination of endothelial marker targeting and the use of shear stress-controlled nanoparticle capture can be used for selective intratumoral drug delivery.

Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.

Background: Uterine sarcoma is an aggressive malignancy with a poor prognosis. This study aimed to determine the expression of CD146, P53, and Ki-67 in uterine sarcoma and to evaluate their prognostic significance., Methods: We retrospectively analyzed the prognosis and clinicopathologic features of 68 patients with uterine sarcoma. Immunohistochemical analyses of CD146, P53, and Ki-67 were performed in tissue samples collected from these patients and their relationship with prognosis was investigated., Results: The 5-year overall survival (OS) rate was 46 %. Endometrial stromal sarcoma (ESS) patients had a better prognosis than leiomyosarcoma (LMS) patients, with a 2-year survival rate of 82 %. The membrane and cytoplasm of tumor cells exhibited CD146 overexpression in 8 (32 %) ESS cases, which was less than the 25 (69.4 %) cases observed in LMS and 2 (28.6 %) in MMMT. CD146 overexpression in the membrane and cytoplasm of tumor cells was closely related to lymph node metastasis (P = 0.021) and Ki-67 overexpression (P = 0.0053); there was no significant correlation with age, tumor size, International Federation of Obstetrics and Gynecology stage, or P53 overexpression in LMS., Conclusions: CD146, P53, and Ki-67 are overexpressed in uterine sarcoma. CD146 expression correlates with lymph node metastasis and is associated with poor OS in LMS; it may be a potential prognostic marker for LMS.

An ultrasensitive electrochemical immunosensor for the detection of cluster of differentiation 146 antigen (CD146) based on TiO2 colloidal sphere laden Au/Pd nanoparticles (Au/Pd@TiO2) was developed. In this work, reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) was applied as an electrode modifying material to modify the surface of a glassy carbon electrode (GCE). Au/Pd@TiO2 was used as the secondary-antibody (Ab2) label for the fabrication of the immunosensor. Amperometric response of the immunosensor for electrocatalytic reduction of hydrogen peroxide (H2O2) was recorded. Electrochemical impedance spectroscopy (EIS) proved that fabrication of the immunosensor was successful. The anti-CD146 primary antibody (Ab1) was immobilized on the rGO-TEPA modified GCE by a cross-linking reagent of glutaraldehyde (GA). With Ab1 immobilized onto the rGO-TEPA modified GCE and Ab2 linked with Au/Pd@TiO2, the immunosensor displayed a wide linear range (0.0050-20 ng mL(-1)), a low detection limit (1.6 pg mL(-1)), good reproducibility, good selectivity and acceptable stability. The designed sensing strategy may provide a potential application in the detection of other tumor markers.

A subset of T cells defined by the cell surface expression of MCAM (CD146) has been identified in the peripheral circulation of healthy individuals. These cells comprise approximately 3% of the pool of circulating T cells, have an effector memory phenotype, and are capable of producing several cytokines. Notably, the MCAM positive cells are enhanced for IL-17 production compared to MCAM negative effector memory T cells. These cells are committed to IL-17 production and do not require in vitro polarization with exogenous cytokines. MCAM positive T cells also demonstrate an increased ability to bind to endothelial monolayers. In numerous autoimmune diseases these cells are found at increased proportions in the peripheral circulation, and at the sites of active inflammation in patients with autoimmune disease, these cells appear in large numbers and are major contributors to IL-17 production. Studies to date have been performed with human subjects and it is uncertain if appropriate mouse models exist for this cell type. These cells could represent early components of the adaptive immune response and serve as targets of therapy in these diseases, although much work remains to be performed in order to discern the exact nature and function of these cells., (Published by Elsevier B.V.)

Purpose: Nano dense-silica (dSiO2) has many advantages such as adjustable core-shell structure, multiple drug delivery, and controllable release behavior. Improving the gastric tumor-specific targeting efficiency based on the development of various strategies is crucial for anti-cancer drug delivery systems., Methods: Superparamagnetic iron oxide nanoparticles (SPION) were coated with dSiO2 as core-shell nanoparticles, and labeled with near infra-red fluorescence (NIRF) dye 800ZW (excitation wavelength: 778 nm/emission wavelength: 806 nm) and anti-CD146 monoclonal antibody YY146 for magnetic resonance (MR)/NIRF imaging study in xenograft gastric cancer model. The morphology and the size of pre- and postlabeling SPION@dSiO2 core-shell nanoparticles were characterized using transmission electron microscopy. Iron content in SPION@dSiO2 nanoparticles was measured by inductively coupled plasma optical emission spectrometry. Fluorescence microscopy and fluorescence-activated cell sorter studies were carried out to confirm the binding specificity of YY146 and 800ZW-SPION@dSiO2-YY146 on MKN45 cells. In vivo and in vitro NIRF imaging, control (nanoparticles only) and blocking studies, and histology were executed on MKN45 tumor-bearing nude mice to estimate the affinity of 800ZW-SPION@dSiO2-YY146 to target tumor CD146., Results: 800ZW-SPION@dSiO2-YY146 nanoparticles were uniformly spherical in shape and dispersed evenly in a cell culture medium. The diameter of the nanoparticle was 20-30 nm with 15 nm SPION core and ~10 nm SiO2 shell, and the final concentration was 1.7 nmol/mL. Transverse relaxivity of SPION@dSiO2 dispersed in water was measured to be 110.57 mM(-1)·s(-1). Fluorescence activated cell sorter analysis of the nanoparticles in MKN45 cells showed 14-fold binding of 800ZW-SPION@dSiO2-YY146 more than the control group 800ZW-SPION@dSiO2. Series of NIRF imaging post intravenous injection of 800ZW-SPION@dSiO2-YY146 demonstrated that the MKN45 xenograft tumor model could be clearly identified as early as a time point of 30 minutes postinjection. Quantitative analysis revealed that the tumor uptake peaked at 24 hours postinjection., Conclusion: This is the first successful study of functional nanoparticles for MR/NIRF imaging of cell surface glycoprotein CD146 in gastric cancer model. Our results suggest that 800ZW-SPION@dSiO2-YY146 nanoparticles will be applicable in tumor for image-guided therapy/surgery.

The circulating endothelial progenitor cells (EPCs) in blood of acute myocardial infarction (AMI) patient have been monitored in many previous studies. The number of circulating EPC increases in the blood of patients at onset of the AMI. EPC is originated from bone marrow. It performs vessel regeneration. There are many markers used for detecting EPC. Four of these markers, CD31, CD34, CD45, and CD146, were concurrently detected at the single cell level for the identification of EPC in the present preliminary study. The CD45 negative cell sorting was performed to peripheral blood mononuclear cells (PBMCs) acquired from four AMI patients with a magnetic bead sorter, since, EPCs expressed CD45 negative or dim. The resultant PBMC eluents were treated with quantum-antibody conjugates for the probing four different markers of EPCs and then applied to a high-content single cell imaging cytometer using acousto-optical tunable filter (AOTF). The use of quantum dot, with narrow emission wavelength range and AOTF enabling cellular image at a particular single wavelength, is very advantageous for accurate high-content AMI diagnosis based on simultaneous monitoring of many markers. The number of EPC increased as compared with control in three of four AMI patients. In this approach, two EPC subtypes were found, CD31(+), CD34(+), CD45(-/dim), CD146(-) as early outgrowth EPCs and CD31(+), CD34(+), CD45(-/dim), CD146(+) as late outgrowth EPCs. Patient 1 had CD31(+), CD34(+), CD45(-/dim), CD146(+) cells whose percentage was 4.21% of cells. Patient 2 had 2.38% of CD31(+), CD34(+), CD45(-/dim), CD146(-) cells and patient 3 had 4.28% of CD31(+), CD34(+), CD45(-/dim), CD146(+) cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Circulating endothelial cells (CECs) have been proposed as a biomarker for the assessment of patients with solid tumors. However, few data are available in non-small cell lung cancer (NSCLC). We therefore analyzed the clinical significance of CECs in newly diagnosed NSCLC patients. In addition, we tried to determine the prognostic value of CECs in NSCLC., Methods: In this prospective study, 151 newly diagnosed NSCLC patients and 25 healthy volunteers were included. Furthermore, 25 patients with a partial response (n=15) or stable disease (n=10) after treatment were evaluated at recurrence with a mean follow-up of 117 days (range: 47-364 days). CECs were counted using magnetic beads coupled to a specific antibody against CD146., Results: The pre-treatment CEC count was significantly higher in patients with all histological subtypes of NSCLC than in healthy volunteers (p&lt;0.0001). High baseline CEC counts were significantly correlated with advanced clinical stages (p=0.026), weight loss (p=0.03), and poorly differentiated NSCLC (p=0.02). The amount of CECs increased significantly at recurrence compared with their amount after treatment in 20/21 assessable patients (p=0.0001). Nevertheless, there was no significant correlation between baseline CEC count and median duration of progression-free survival (p=0.402)., Conclusions: Increased CEC counts were present in patients with newly diagnosed NSCLC compared with healthy subjects. Elevated levels of baseline CECs correlated with high-risk factors in NSCLC. In addition, increased CEC count during follow-up seems to be correlated with recurrence in NSCLC patients.

In vitro expanded bone marrow stromal cells contain at least two populations of fibroblasts, a CD146/MCAM positive population, previously reported to be critical for establishing the stem cell niche and a CD146-negative population that expresses CUB domain-containing protein 1 (CDCP1)/CD318. Immunohistochemistry of marrow biopsies shows that clusters of CDCP1+ cells are present in discrete areas distinct from areas of fibroblasts expressing CD146. Using a stromal cell line, HS5, which approximates primary CDCP1+ stromal cells, we show that binding of an activating antibody against CDCP1 results in tyrosine-phosphorylation of CDCP1, paralleled by phosphorylation of Src Family Kinases (SFKs) Protein Kinase C delta (PKC-δ). When CDCP1 expression is knocked-down by siRNA, the expression and secretion of myelopoietic cytokines is increased. These data suggest CDCP1 expression can be used to identify a subset of marrow fibroblasts functionally distinct from CD146+ fibroblasts. Furthermore the CDCP1 protein may contribute to the defining function of these cells by regulating cytokine expression.

The in vivo identity of stem cells is not yet clear. Numerous studies involve the perivascular niches as providers of stem cells during regenerative processes. CD146, in humans, as well as gicerin, at chicken, play roles in neuronal development and neurites extension. CD146 is a marker of stemness but also a pericytary marker. Stem cells in vascular niches can differentiate in neural cells. By applying CD146 and CD105 antibodies on human retinas from glaucomatous eyes, CD146-positive retinal ganglion cells (RGCs) were found, some being placed in perivascular positions; ongoing processes of neurites extension were related to these neurons. On other hand, RGCs were positively labeled by CD105 antibodies. These results support the hypothesis that in glaucoma eyes the CD146-positive RGCs result from regenerative processes driven by stem cells in the retinal perivascular niches. Further experiments are needed to evaluate whether CR146-positive neurons indicate also a physiological process of maintenance of retina., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hepatocellular carcinomas are well-vascularized tumors; the endothelial cells in these tumors have a specific phenotype. Our aim was to develop a new approach for tumor-specific drug delivery with monoclonal antibody targeting of endothelial ligands. CD146, a molecule expressed on the endothelial surface of hepatocellular carcinoma, was identified as a promising candidate for targeting. In the present study, endothelial cells immediately captured circulating anti-CD146 (ME-9F1) antibody, while antibody binding in tumors was significantly higher than in hepatic endothelium. Macroscopically, after intravenous injection, there were no differences in the mean accumulation of anti-CD146 antibody in tumor compared to liver tissue , due to a compensating higher blood vessel density in the liver tissue. Additional blockade of nontumoral epitopes and intra-arterial administration, improved selective antibody capture in the tumor microvasculature and largely prevented antibody distribution in the lung and liver. The potential practical use of this approach was demonstrated by imaging of radionuclide-labeled ME-9F1 antibody, which showed excellent tumor-selective uptake. Our results provide a promising principle for the use of endothelial markers for intratumoral drug delivery. Tumor endothelium-based access might offer new opportunities for the imaging and therapy of hepatocellular carcinoma and other liver malignancies.

The focus of this study is the characterization of human T cell blood-brain barrier migration and corresponding molecular trafficking signatures. We examined peripheral blood and cerebrospinal fluid immune cells from patients under long-term anti-very late antigen-4 (VLA-4)/natalizumab therapy (LTNT) and from CNS specimens. LTNT patients' cerebrospinal fluid T cells exhibited healthy central-/effector-memory ratios, but lacked CD49d and showed enhanced myeloma cell adhesion molecule (MCAM) expression. LTNT led to an increase of PSGL-1 expression on peripheral T cells. Although vascular cell adhesion molecule-1 (VLA-4 receptor) was expressed at all CNS barriers, P-selectin (PSGL-1-receptor) was mainly detected at the choroid plexus. Accordingly, in vitro experiments under physiological flow conditions using primary human endothelial cells and LTNT patients' T cells showed increased PSGL-1-mediated rolling and residual adhesion, even under VLA-4 blockade. Adhesion of MCAM(+)/TH17 cells was not affected by VLA-4 blocking alone, but was abrogated when both VLA-4 and MCAM were inhibited. Consistent with these data, MCAM(+) cells were detected in white matter lesions, and in gray matter of multiple sclerosis patients. Our data indicate that lymphocyte trafficking into the CNS under VLA-4 blockade can occur by using the alternative adhesion molecules, PSGL-1 and MCAM, the latter representing an exclusive pathway for TH17 cells to migrate over the blood-brain barrier., (© 2014 Schneider-Hohendorf et al.)

Interleukin-17 (IL-17) has been associated with the pathogenesis of numerous autoimmune diseases. CD4+ T cells secreting IL-17 are termed Th17 cells. CD8+ T cells, designated Tc17 cells, are also capable of secreting IL-17. Here we describe a population of Tc17 cells characterized by the expression of surface CD146, an endothelial adhesion molecule. These cells display signatures of a human Tc17 genotype and phenotype. Circulating CD8+CD146+ T cells are present in low levels in healthy adults. Elevations in CD8+CD146+ T cells are found in Behcet's disease and birdshot retinochoroidopathy, which have been reported to have HLA class I associations. Sarcoidosis does not have a class I association and displays an increase in CD4+ CD146+ T cells but not in CD8+CD146+ T cells. CD146 on these cells may facilitate their ability to bind to, and migrate through, endothelium, as has been reported for CD4+CD146+ T cells., (Published by Elsevier Inc.)

The ability to selectively block the entry of leukocytes into the central nervous system (CNS) without compromising the immune system is an attractive therapeutic approach for treating multiple sclerosis (MS). Using endothelial CD146-deficienct mice as a MS model, we found that endothelial CD146 plays an active role in the CNS-directed extravasation of encephalitogenic T cells, including CD146(+) TH1 and TH17 lymphocytes. Moreover, treating both active and passive MS models with the anti-CD146 antibody AA98 significantly decreased the infiltrated lymphocytes in the CNS and decreased neuroinflammation. Interestingly, the ability of AA98 to inhibit the migration of CD146(+) lymphocytes was dependent on targeting endothelial CD146, but not lymphocytic CD146. These results suggest a key molecular target located on the blood-brain barrier endothelium that mediates the extravasation of inflammatory cells into the CNS. In addition, our data suggest that the AA98 is a promising candidate for treating MS and other CNS autoimmune diseases.

Background: Endothelial damage is a critical step in the development of (xeno) transplantation-related and cardiovascular pathology. In humans, the amount of circulating endothelial cells (CEC) correlates to disease intensity and functions as a valuable damage marker. While (xeno) transplantation and cardiovascular research is regularly performed in porcine models, the paucity of antibodies against porcine endothelium epitopes hinders the use of CEC as damage marker., Objective: This study aimed to develop a method for porcine CEC detection using anti-human antibodies against porcine endothelium epitopes., Methods: Human umbilical vein endothelial cells (HUVEC, control) and their swine equivalent (SUVEC) were used to assess the cross-species immunoreactivity of fluorescently labeled anti-human CD31/CD51/CD54/CD62E/CD105/CD106/CD144/CD146/PAL-E/lectin-1/vWF antibodies by isotype-controlled fluorescence-activated cell sorting (FACS) and confocal microscopy. Next, reactivity was ascertained with mature porcine kidney-derived endothelial cells (PKEC), and a FACS-based whole blood CEC quantification method was employed using osmotic erythrolysis and CD105 and CD146 double staining after CD45 exclusion., Results: Of the 21 assayed antibodies, the MEM-229 clone of CD105 and P1H12 clone of CD146 showed immunoreactivity with SUVEC and PKEC. Double staining showed baseline porcine CEC count of 673.1 ± 551.4 CEC/ml, while the first 7.5 ml of drawn blood (representative of vascular damage) contained 1118 ± 661.4 CEC/ml (n = 14, P = 0.04). A second experiment (n = 5) including CD45 exclusion identified only 14.5 ± 10.8% double-positive CD105-146 events per ml blood., Conclusion: Porcine endothelium can be specifically labeled using anti-human CD146 and CD105 antibodies. These antibodies can therefore be used for the identification and quantification of CEC in porcine whole blood by FACS after osmotic erythrolysis., (© 2013 John Wiley & Sons A/S.)

Effector memory T (T(EM)) cells are a subpopulation of memory T cells that express receptors mediating migration to inflamed tissues and produce various cytokines. Effector memory T-helper (Th)17 (Th17(EM)) cells are thought to be essential for inflammation in Th17-mediated diseases, but have not been studied in detail. To identify superior surface markers to isolate a homogeneous population of Th17(EM) cells from peripheral blood, CD4(+) T cells were isolated from the peripheral blood of healthy donors based on the expression of CCR7, CCR6 and CD146 using six-color flow cytometry. After 4days of culture in the presence of anti-CD3/28 beads, intracellular cytokines were determined by flow cytometric analysis. To investigate the relevance of Th17(EM) cells in Th17-mediated disease, the frequencies of T(EM) -cell subsets in psoriasis were quantified using six-color flow cytometry. An enzyme-linked immunosorbent assay was performed to confirm the interleukin (IL)-17-producing capacity of T(EM) -cell subsets from the peripheral blood of a patient with psoriasis. CCR6(+) CD146(+) T(EM) (CD4(+) CD45RA(-) CCR7(-)) cells had a greater capacity to produce IL-17 than CCR6(+) CD146(-) or CCR6(-) CD146(+) T(EM) cells. Although the percentage of CCR6(+) CD146(+) cells in T(EM) cells was not significantly different between patients with psoriasis and controls, three of eight patients had a higher percentage of CCR6(+) CD146(+) T(EM) cells than the mean +5 standard deviations of the controls. Coexpression of CCR6 and CD146 is a useful marker for Th17(EM) cells. Increasing the number of CCR6(+) CD146(+) Th17(EM) cells in peripheral blood may facilitate estimation of systemic Th17-cell activity in Th17-mediated diseases., (© 2012 Japanese Dermatological Association.)

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions., (Copyright © 2011 Wiley Periodicals, Inc.)

Th17 cells are a subset of CD4(+) T cells characterized by production of IL-17 and are known to be key participants in inflammatory reactions and various autoimmune diseases. In this study we found that a subset of human CD4(+) T cells expressing MCAM (CD146) have higher mRNA levels of RORC2, IL-23R, IL-26, IL-22, IL-17A, but not IFN-γ, compared to CD4(+) T cell not expressing CD146. Upon TCR stimulation with CD3/CD28, CD4(+)CD146(+) T cells secrete significantly more IL-17A, IL-6, and IL-8 than do CD4(+)CD146(-) T cells. Low frequencies of CD4(+)CD146(+) T cells are found in the circulation of healthy adults, but the frequency of these cells is significantly increased in the circulation of patients with inflammatory autoimmune diseases including Behcet's, sarcoidosis and Crohn's disease. Patterns of gene expression and cytokine secretion in these cells are similar in healthy and disease groups. In Crohn's disease, the increase in CD4(+)CD146(+) cells in the circulation correlates with disease severity scores. These data indicate that expression of CD146 on CD4(+) T cells identifies a population of committed human Th17 cells. It is likely the expression of CD146, an endothelial adhesion molecule, facilitates adherence and migration of Th17 cells through the endothelium to sites of inflammation., (Published by Elsevier Ltd.)

To date, in lung cancer, early attempts to modulate the immune system via vaccine-based therapeutics have been unsuccessful. An improved understanding of tumor immunology has facilitated the production of more sophisticated lung cancer vaccines. It is anticipated that it will likely require multiple epitopes of a diverse set of genes restricted to multiple haplotypes to generate a truly effective vaccine that is able to overcome the various immunologic escape mechanisms that tumors employ. Other issues to overcome include optimal patient selection, which adjuvant agent to use, and how to adequately monitor for an immunologic response. This review discusses the most promising vaccination strategies for non-small cell lung cancer including the allogeneic tumor cell vaccine belagenpumatucel-L, which is a mixture of 4 allogeneic non-small cell lung cancer cell lines genetically modified to secrete an antisense oligonucleotide to transforming growth factor β2 and 3 other target protein-specific vaccines designed to induce responses against melanoma-associated antigen A3, mucin 1, and epidermal growth factor.

Objectives: We previously reported a correlation between levels of micro particles carrying CD31 (PMP(CD31+)) and disease activity in MS. However, the effects of long term (12 month) treatment with high dose, high frequency interferon-β1a (Rebif™) on plasma levels of PMP(CD31+), PMP(CD146+), and PMP(CD54+) and MRI measures of disease activity have not yet been assessed., Methods: During this prospective 1-year study, we used flow cytometry to measure changes in plasma micro particles (PMP) bearing CD31 (PMP(CD31+)), CD146 (PMP(CD146+)), and CD54/ICAM-1 (PMP(CD54+)) in 16 consecutive patients with relapsing-remitting MS (RRMS) before and after 3, 6, and 12 months of subcutaneous therapy with interferon-beta1a (44 micrograms, 3X weekly). At each visit, clinical exams and expanded disability status scale (EDSS) scores were recorded., Results: Plasma levels of PMP(CD31+), and PMP(CD54+) were significantly reduced by treatment with IFN-β1a. PMP(CD146+) appeared to decrease only at 3 months and did not persist at 6 and 12 months (p = 0.0511). In addition, the decrease in plasma levels of PMP(CD31+) and PMP(CD54+) levels at 12 months were associated with a significant decrease in the number and volume of contrast enhancing T1-weigthed lesions., Conclusion: Our data suggest that serial measurement of plasma micro particles (PMP), particularly in the initial stages of MS (when neuro-inflammatory cascades are more intense), may serve as reliable and reproducible surrogate markers of response to IFN-β1a therapy for MS. In addition, the progressive decline in plasma levels of PMP(CD31+) and PMP(CD54+) further supports the concept that IFN-β1a exerts stabilizing effect on the cerebral endothelial cells during pathogenesis of MS.

Background: Conflicting research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as both a tumor promoter and a tumor suppressor in the development of breast cancer. To resolve this, we have re-investigated the role of this CAM in the progression of human breast cancer cells., Methods: Three breast cancer cell lines were used for the tests: one luminal-like breast cancer cell line, MCF7, which did not express any METCAM/MUC18, and two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which expressed moderate levels of the protein.MCF7 cells were transfected with the human METCAM/MUC18 cDNA to obtain G418-resistant clones which expressed the protein and were used for testing effects of human METCAM/MUC18 expression on in vitro motility and invasiveness, and in vitro and in vivo tumorigenesis. Both MDA-MB-231 and MDA-MB-468 cells already expressed METCAM/MUC18. They were directly used for in vitro tests in the presence and absence of an anti-METCAM/MUC18 antibody., Results: In MCF7 cells, enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, anchorage-independent colony formation (in vitro tumorigenesis), and in vivo tumorigenesis. In both MDA-MB-231 and MDA-MB-468 cells, the anti-METCAM/MUC18 antibody inhibited both motility and invasiveness. Though both MDA-MB-231 and MDA-MB-468 cells established a disorganized growth in 3D basement membrane culture assay, the introduction of the anti-METCAM/MUC18 antibody completely destroyed their growth in the 3D culture., Conclusion: These findings support the notion that human METCAM/MUC18 expression promotes the progression of human breast cancer cells by increasing their motility, invasiveness and tumorigenesis.

Blastocyst embedding is very similar to neoplasm invasion. Blastocyst embedding is seeding the young plant of life, while neoplasm invasion is knocking at the door of death. Overexpression of melanoma cell adhesion molecule (CD146 or MCAM), a novel member of the immunoglobulingene superfamily, promotes invasion, metastasis, growth and survival of malignant cells, and implantation of blastocyst embedding in placenta. We hypothesize that CD146 may be a key gene both in neoplasm invasion and blastocyst embedding because of its ability in regulating cell invasion. The regulation of CD146 expression may be a control switch in the progress of the neoplasm invasion and blastocyst embedding. If the hypothesis is correct, the inhibition of CD146 can be used to prevent and/or treat tumor invasion. Current treatment modalities of tumor invasion include different therapies: surgical resection, radiotherapy, chemotherapy, etc. These treatments are all non-specific to tumor cells. If further studies proof our hypothesis, CD146 may be a candidate target gene in gene therapy of tumor invasion and in regulation of blastocyst embedding., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: Although histone deacetylase inhibitors (HDACi) are emerging as a new class of anticancer agents, one of the most significant concerns is that interactions with a wide array of substrates using these agents might initiate both therapeutic and undesired protective responses. Here, we sought to identify the potential protective reactions initiated by HDACi and determine whether targeting these reactions would enhance the antitumoral activity of HDACi., Experimental Design: Gene expression profiles were analyzed by cDNA microarray in Molt-4 cells before and after treatment of vorinostat. Induction of CD146 by vorinostat was examined in a wide range of tumors and nonmalignant cells. AA98, an anti-CD146 monoclonal antibody, was used to target CD146 function. Synergistic antitumoral and antiangiogenic effects between AA98 and vorinostat were examined both in vitro and in vivo. The potential effect of combined AA98 and vorinostat treatment on the AKT pathway was determined by Western blotting., Results: The induction of CD146 is a common phenomenon in vorinostat-treated cancer but not in nonmalignant cells. Targeting of CD146 with AA98 substantially enhanced vorinostat-induced killing via the suppression of activation of AKT pathways in cancer cells. Moreover, AA98 in combination with vorinostat significantly inhibited angiogenesis. In vivo, AA98 synergized with vorinostat to inhibit tumor growth and metastasis., Conclusion: The present study provided the first evidence that an undesired induction of CD146 could serve as a protective response to offset the antitumor efficacy of vorinostat. On the other hand, targeting CD146 in combination with vorinostat could be exploited as a novel strategy to more effectively kill cancer cells., (©2010 AACR.)

In ischemic acute kidney injury, renal blood flow is decreased. We have previously shown that reperfused, transplanted kidneys exhibited ischemic injury to vascular endothelium and that preservation of peritubular capillary endothelial integrity may be critical to recovery from ischemic injury. We hypothesized that bone marrow-derived (BMD) endothelial progenitor cells (EPCs) might play an important role in renal functional recovery after ischemia. We tested this hypothesis in recipients of cadaveric renal allografts before and for 2 weeks after transplantation. We found that the numbers of circulating CD34-positive EPCs and CD146-positive endothelial cells (ECs) decreased immediately after ischemia-reperfusion. In renal allograft tissues obtained 1 hr after reperfusion, CD34-positive cells were more frequently observed along the endothelial lining of peritubular capillaries compared with non-ischemic controls. Moreover, 0-17.5% of peritubular capillary ECs were of recipient origin. In contrast, only 0.1-0.7% of tubule cells were of recipient origin. Repeat graft biopsy samples obtained 35 and 73 days after transplant did not contain capillary ECs of recipient origin, whereas 1.4% and 12.1% of tubule cells, respectively, were of recipient origin. These findings suggest that BMD EPCs and ECs may contribute to endothelial repair immediately after ischemia-reperfusion.